Compounds, compositions and methods for preventing neurodegeneration in acute and chronic injuries in the central nervous system

ABSTRACT

The present invention provides compositions and methods for prevention and prophylaxis of neurological diseases accompanied by neuronal death. The invention includes synthesis of 5-benzylamino salicylic acid (BAS) and its derivatives. BAS and its derivatives protect cortical neurons from toxic insults by N-methyl-D-aspartate, Zn 2+ , and reactive oxygen species. Thus, the present invention provides compositions and methods for treating stroke, traumatic brain and spinal cord injury, epilepsy, and neurodegenerative diseases that are accompanied by severe neuronal loss via excitotoxicity, Zn 2+  neurotoxicity, and free radical neurotoxicity.

FIELD OF THE INVENTION

[0001] The present invention is related to novel salicylic compounds,compositions and methods for prevention and prophylaxis of neurologicaldiseases accompanied by neuronal death.

BACKGROUND OF THE INVENTION Excitotoxicity and Brain Diseases

[0002] Excess activation of ionotropic glutamate receptors sensitive toN-methyl-D-aspartate (NMDA receptors) produces neuronal death and hasbeen known to mediate various neurological diseases [Choi, Neuron1:623-634 (1988)]. Glutamate, the excitatory neurotransmitter, ismassively accumulated in brain subjected to hypoxic-ischemic injuries,which activates ionotropic glutamate receptors permeable to Ca²⁺ and Na⁺and then causes neuronal death [Choi and Rothman, Annu Rev Neurosci13:171-182 (1990)]. Antagonists of NMDA receptors remarkably attenuatebrain injury following hypoglycemia, hypoxia, or hypoxic-ischemia[Simon, Swan, Griffiths, and Meldrum. Science 226:850-852 (1984); Park,Nehls, Graham, Teasdale, and McCulloch, Ann Neurol 24:543-551 (1988).;Wieloch, Science 230:681-683 (1985); Kass, Chambers, and Cottrell, Exp.Neurol. 103:116-122 (1989); Weiss, Goldberg, and Choi, Brain Res.380:186-190 (1986)]. Thus, NMDA receptor antagonists possess therapeuticpotentials to protect brain against hypoglycemia, hypoxia, andhypoxic-ischemic injuries.

[0003] Excitotoxicity appears to contribute to neuronal degenerationfollowing traumatic brain injury (TBI). Levels of quinolinic acid, anendogenous agonist of NMDA receptors, was increased 5- to 50-fold inhuman patients with TBI [E. H. Sinz, P. M. Kochanek, M. P. Heyes, S. R.Wisniewski, M. J. Bell, R. S. Clark, S. T. DeKosky, A. R. Blight, and D.W. Marion]. Quinolinic acid is increased in the cerebrospinal fluid andassociated with mortality after TBI in humans [J.Cereb.Blood Flow Metab.18:610-615, (1998)]. In animal models of brain trauma, levels ofglutamate and aspartate were markedly increased [Faden, Demediuk,Panter, and Vink, Science 244:798-800 (1989)]. Glutamate release wasalso observed in rat spinal cord following impact trauma [Demediuk,Daly, and Faden. J Neurochem J. Neurochem. 52 :1529-1536 (1989)]. NMDAreceptor antagonists attenuate neuronal death following traumatic brainor spinal cord injuries [Faden, Lemke, Simon, and Noble. J.Neurotrauma.5:33-45(1988); Okiyama, Smith, White, Richter, and McIntosh.J.Neurotrauma. 14:211-222 (1997)].

[0004] Glutamate plays a central role in the induction and thepropagation of seizures [Dingledine, McBain, and McNamara,Trends.Pharmacol.Sci. 11:334-338 (1990); Holmes. Cleve.Clin.J.Med.62:240-247 (1995)]. NMDA receptor antagonists were shown to act asanticonvulsants and antiepileptogenic drugs in various models ofepilepsy [Anderson, Swartzwelder, and Wilson, J.Neurophysiol. 57:1-21(1987); Wong, Coulter, Choi, and Prince. Neurosci.Lett. 85:261-266(1988); McNamara, Russell, Rigsbee, and Bonhaus, Neuropharmacology27:563-568 (1988)].

[0005] Amyotrophic lateral sclerosis (ALS) is accompanied bydegeneration of both upper and lower motor neurons and marked byneurogenic atrophy, weakness, and fasciculation. While the pathogenesisof ALS remains to be resolved, excitotoxicity has been expected toparticipate in the process of ALS. In particular, ALS patients showincreased levels of extracellular glutamate and defects in glutamatetransport. Administration of excitotoxins mimicked pathological changesin the spinal cord of ALS patients [Rothstein. Clin.Neurosci. 3:348-359(1995); Ikonomidou, Qin, Labruyere, and Olney J.Neuropathol.Exp.Neurol.55:211-224 (1996)].

[0006] Antagonizing NMDA receptors appears to be applied to treatParkinson's disease (PD). Several antagonists of NMDA receptors protectdopaminergic neurons from the neurotoxin MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) [Lange, Loschmann, Sofic,Burg, Horowski, Kalveram, Wachtel, and Riederer. Naunyn SchmiedebergsArch.Pharmacol. 348:586-592 (1993); Brouillet and Beal. Neuroreport.4:387-390 (1993)]. NMDA receptor antagonists also amelioratelevodopa-induced dyskinesia and thus can improve the therapeutic effectsof levodopa [Papa and Chase, Ann.Neurol. 39:574-578 (1996); Marin, Papa,Engber, Bonastre, Tolosa, and Chase, Brain Res. 736:202-205 (1996)]. TwoNMDA receptor antagonists, memantine and dextromethophan, have beenproved beneficial in treating PD patients [Verhagen, Del Dotto, Natte,van den Munckhof, and Chase, Neurology 51:203-206 (1998); Merello,Nouzeilles, Cammarota, and Leiguarda. Clin.Neuropharmacol. 22:273-276(1999)].

[0007] Huntington's disease (HD) is a progressive neurodegenerativedisease predominantly affecting small- and medium-sized inteneurons butsparing NADPH-diaphorase neurons containing somatostatin andneuropeptide in the striata. These pathological features of HD areobserved in the striatal tissues following the intrastriatal injectionsof quinolinic acid or cultured striatal neurons exposed to NMDA, raisingthe possibility that NMDA receptor-mediated neurotoxicity contributes toselective neuronal death in HD [Koh, Peters, and Choi, Science 234:73-76(1986); Beal, Kowall, Ellison, Mazurek, Swartz, and Martin, Nature321:168-171 (1986); Beal, Ferrante, Swartz, and Kowall. J.Neurosci.11:1649-1659 (1991)].

Free Radicals and Brain Diseases

[0008] Free radicals are produced in degenerating brain areas followinghypoxic-ischemia or traumatic brain and spinal cord injuries [Hall andBraughler, Free Radic.Biol.Med. 6:303-313 (1989); Anderson and Hall,Ann.Emerg.Med. 22:987-992 (1993); Siesjo and Siesjo, Eur.J.Anaesthesiol.13:247-268(1996); Love, Brain Pathol. 9:119-131 (1999)]. Antioxidants ormaneuvers scavenging free radicals attenuate brain damages byhypoxic-ischemia or traumatic injuries [Faden, Pharmacol.Toxicol.78:12-17 (1996); Zeidman, Ling, Ducker, and Ellenbogen, J.Spinal.Disord.9:367-380 (1996); Chan, Stroke 27:1124-1129 (1996); Hall,Neurosurg.Clin.N.Am. 8:195-206 (1997)]. Extensive evidence supports thatfree radicals can be produced in brain areas undergoing degeneration inneurodegenerative diseases possibly due to point mutations in Cu/Znsuperoxide dismutase in ALS, decreased glutathione and increased iron inPD, accumulation of iron in AD, or mitochondrial dysfunction in HD[Rosen, Siddique, Patterson, Figlewicz, Sapp, Hentati, Donaldson, Goto,O'Regan, and Deng. Nature 362:59-62 (1993); Jenner and Olanow, Neurology47:S161-S170 (1996); Smith, Harris, Sayre, and Perry,Proc.Natl.Acad.Sci.U.S.A. 94:9866-9868 (1997); Browne, Ferrante, andBeal, Brain Pathol. 9:147-163 (1999)]. Accordingly, antioxidants havebeen neuroprotective against such neurodegenerative diseases [Jenner,Pathol. Biol. (Paris.) 44:57-64 (1996); Beal, Ann. Neurol. 38:357-366(1995); Prasad, Cole, and Kumar. J.Am.Coll.Nutr. 18:413-423 (1999);Eisen and Weber, Drugs Aging 14:173-196 (1999); Grundman,Am.J.Clin.Nutr. 71:630S.-636S (2000)].

Zinc and Brain Diseases

[0009] Zn²⁺ mediates neurodegenerative process observed in seizure,ischemia, trauma, and Alzheimers disease (AD). The centraladministration of kainate, a seizure-inducing excitotoxin, causes thetranslocation of Zn²⁺ into postsynaptic degenerating neurons in severalforebrain areas [Frederickson, Hernandez, and McGinty. Brain Res.480:317-321 (1989)]. Blockade of Zn²⁺ translocation with Ca-EDTAattenuates neuronal loss following a transient forebrain ischemia ortraumatic brain injury [Koh, Suh, Gwag, He, Hsu and Choi, Science 272:1013-1016 (1996); Suh, Chen, Motamedi, Bell, Listiak, Pons, Danscher,and Frederickson, Brain Res. 852 :268-273 (2000)]. Zn²⁺ is observed inthe extracellular plaque and degenerating neurons in AD, which likelycontributes to neuronal degeneration in AD [Bush, Pettingell, Multhaup,Paradis, Vonsattel, Gusella, Beyreuther, Masters, and Tanzi, Science265:1464-1467 (1994); Suh, Jensen, Jensen, Silva, Kesslak, Danscher, andFrederickson. Brain Res. 852:274-278 852 (2000)].

SUMMARY OF THE INVENTION

[0010] The present invention provides novel 5-benzylamino salicylic acid(BAS) and its derivatives represented by the following formula (I):

[0011] wherein,

[0012] X is CO, SO₂ or (CH₂)_(n) (where n is an integer of 1 to 5,inclusive);

[0013] R₁ is hydrogen, alkyl or alkanoyl;

[0014] R₂ is hydrogen or alkyl;

[0015] R₃ is hydrogen or an acetoxy group; and

[0016] R₄ is phenyl group which is unsubstituted or substituted with oneor more of the group consisting of nitro, halogen, haloalkyl, and C₁-C₅alkoxy; or a pharmaceutically-acceptable salt thereof.

[0017] The present invention also provides method for protecting centralneurons from acute or chronic injuries to central nervous system (CNS),comprising administering to a patient or a mammal suffering from suchCNS injuries a therapeutically appropriate amount of a neuroprotectivecompound represented by Formula (I).

[0018] The present invention still provides a composition for protectingcentral neurons from acute or chronic injuries to central nervous systemcomprising a neuroprotective compound represented by Formula (I) in atherapeutically appropriate amount.

[0019] The present invention still provides a method for treating orpreventing neurological diseases linked to NMDA neurotoxocity, Zn²⁺neurotoxicity or oxidative stress, comprising administering to a patientor a mammal suffering from such diseases a therapeutically effectiveamount of the compound represented by Formula (I).

[0020] The present invention more provides a use of the compound ofFormula (I) in the manufacture of medicaments for protecting centralneurons from acute or chronic injuries to central nervous system (CNS).

[0021] The above and other features of the present invention will beapparent to those of ordinary skill in the art from the detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is a result testing neuroprotective effects of 5-aminosalicylic acid (AS) against neuronal death induced by an excitotoxinNMDA (1 a), a free radical-producing agent Fe2+ (1 b) or Zn2+ (1 c) incultured cortical cells.

[0023]FIG. 2 is a result testing neuroprotective effects of5-benzylamino salicylate (BAS) against neuronal death induced by NMDA (2a), Fe2+ (2 b), buthionine sulfoximine (BSO) (2 c) or Zn2+ (2 d) incultured cortical cells.

[0024]FIG. 3 is a result testing neuroprotective effects of5-(4-nitrobenzyl)aminosalicylic acid (NBAS) against neuronal deathinduced by NMDA (3 a), Fe2+ (3 b) or Zn2+ (3 c) in cultured corticalcells.

[0025]FIG. 4 is a result testing neuroprotective effects of5-(4-chlorobenzyl)aminosalicylic acid (CBAS) against neuronal deathinduced by NMDA (4 a), Fe2+ (4 b) or Zn2+ (4 c) in cultured corticalcells.

[0026]FIG. 5 is a result testing neuroprotective effects of5-(4-Trifluoromethylbenzyl)aminosalicylic acid (TBAS) against neuronaldeath mediated by NMDA (5 a), Fe2+ (5 b) or Zn2+ (5 c) in culturedcortical cells.

[0027]FIG. 6 is a result testing neuroprotective effects of5-(4-Fluorobenzyl)aminosalicylic acid (FBAS) against neuronal deathinduced by NMDA (6 a) or Fe2+ (6 b) in cultured cortical cells.

[0028]FIG. 7 is a result testing neuroprotective effects of5-(4-methoxybenzyl)aminosalicylic acid(MBAS) against neuronal deathinduced by NMDA (7 a) or Fe2+ (7 b) in cultured cortical cells.

[0029]FIG. 8 is a result testing neuroprotective effects of5-(pentafluorobenzyl)amino salicylic acid (PBAS) against neuronal deathinduced by NMDA (8 a), Fe2+ (8 b), BSO (8 c) or Zn2+ (8 d) in culturedcortical cells.

[0030]FIG. 9 is a result testing neuroprotective effects ofethyl-5-(4-nitrobenzyl)amino-2-hydroxy ethylbenzoate (NAHE) againstneuronal death induced by NMDA (9 a) or Fe2+ (9 b) in cultured corticalcells.

[0031]FIG. 10 is a result testing neuroprotective effects of5-(4-nitrobenzyl)-N-acetylamino-2-hydroxy ethylbenzoate (NNAHE) againstneuronal death induced by NMDA (10 a) or Fe2+ (10 b) in culturedcortical cells.

[0032]FIG. 11 is a result testing neuroprotective effects of5-(4-nitrobenzyl)-N-acetylamino-2-acetoxy ethylbenzoate (NNAAE) againstneuronal death induced by NMDA (11 a) or Fe2+ (11 b) in culturedcortical cells.

[0033]FIG. 12 is a result testing neuroprotective effects of5-(4-nitrobenzonyl)aminosalicylic acid(NBAA) against neuronal deathinduced by NMDA (12 a) or Fe2+ (12 b) in cultured cortical cells.

[0034]FIG. 13 is a result testing neuroprotective effects of5-(4-nitrobenzenesulfonyl)aminosalicylic acid (NBSAA) against neuronaldeath induced by NMDA (13 a) or Fe2+ (13 b) in cultured cortical cells.

[0035]FIG. 14 is a result testing neuroprotective effects of5-[2-(4-nitrophenyl)-ethyl]aminosalicylic acid (NPAA) against neuronaldeath induced by NMDA (14 a) or Fe2+ (14 b) in cultured cortical cells.

[0036]FIG. 15 is a result testing neuroprotective effects of5-[3-(4-nitrophenyl)-n-propyl]aminosalicylic acid (NPPAA) againstneuronal death induced by NMDA (15 a) or Fe2+ (15 b) in culturedcortical cells.

DETAILED DESCRIPTION OF THE INVENTION

[0037] We have synthesized 5-benzylamino salicylic acid (BAS) and itsderivatives and demonstrated that these synthetic compounds havemultiple neuroprotective action. First, BAS and its derivativesattenuate NMDA neurotoxicity at doses of 100-1,000 uM. Second, BAS andits derivatives are antioxidants and block free radical neurotoxicity atdoses of 1-300 uM. Finally, BAS and its derivatives attenuate Zn²⁺neurotoxicity. These novel and multiple neuroprotective effects of BASand its derivatives are merited to treat stroke, traumatic brain andspinal cord injury, epilepsy, and neurodegenerative diseases that areaccompanied by excitotoxicity, Zn²⁺ neurotoxicity, and free radicalneurotoxicity.

[0038] The BAS and its derivatives may be synthesized from5-aminosalicylic acid by reacting it with an appropriate compound. Thefollowing reaction schemes illustrate the synthesis of BAS and itsderivatives.

[0039] Reagents (a): Benzyl bromide, 4-nitrobenzyl bromide,4-chlorobenzyl chloride, 4-(trifluoro methyl)benzyl chloride,4-fluorobenzyl bromide, 4-methoxybenzyl chloride, N,N-dimethyl foramide(DMF), triethylamine, room temperature, 4 hr.

[0040] A preferred class of compounds within Formula (I) comprises thosecompounds wherein X is CO, SO₂ or (CH₂)_(n) (where n is an integer of1-5, inclusive); R₁ is hydrogen, C₁-C₅ alkyl or C₂-C₅ alkanoyl; R₂ ishydrogen or C₁-C₅ alkyl; R₃ is hydrogen or an acetoxy group; and R₄ isphenyl group which is unsubstituted or substituted with one or moreselected from the group consisting of nitro, halogen, haloalkyl, andC₁-C₅ alkoxy; or a pharmaceutically-acceptable salt thereof.

[0041] Reagents (a): N,N-dimethylforamide (DMF), triethylamine, roomtemperature, 4 hr.

[0042] Reagents (a) EtOH, H₂SO₄, reflux, 6 hr.

[0043] (b) Acetic anhydride, MeOH, 0° C., 30 min.

[0044] (c) Acetic anhydride, H₂SO₄, 0° C., 30 min.

[0045] A more preferred class of compounds within Formula (I)encompasses those compounds wherein X is CO, SO₂ or (CH₂)_(n) (wheren=1,2,3); R₁ is hydrogen, C₁-C₃ alkyl or C₂-C₃ alkanoyl; R₂ is hydrogenor C₁-C₃ alkyl; R₃ is hydrogen or an acetoxy group; R₄ is phenyl groupwhich is unsubstituted or substituted with one or more selected from thegroup consisting of nitro, halogen, halo(C₁-C₃)alkyl and C₁-C₃ alkoxy;or a pharmaceutically-acceptable salt thereof.

[0046] Reagents (a) N,N-dimethylforamide (DMF), triethylamine, roomtemperature, 4 hr.

[0047] Specific compounds of interest within Formula (I) are as follows:

[0048] 5-benzylaminosalicylic acid (BAS),

[0049] 5-(4-nitrobenzyl)aminosalicylic acid (NBAS),

[0050] (5-(4-chlorobenzyl)aminosalicylic acid (CBAS),

[0051] (5-(4-trifluoromethylbenzyl)aminosalicylic acid (TBAS),

[0052] (5-(4-fluorobenzyl)aminosalicylic acid (FBAS),

[0053] 5-(4-methoxybenzyl)aminosalicylic acid (MBAS)

[0054] 5-(pentafluorobenzyl)aminosalicylic acid (PBAS),

[0055] 5-(4-nitrobenzyl)amino-2-hydroxy ethylbenzoate(NAHE),

[0056] 5-(4-nitrobenzyl)-N-acetylamino-2-hydroxy ethylbenzoate(NNAHE),

[0057] 5-(4-nitrobenzyl)-N-acetylamino-2-acetoxy ethylbenzoate(NNAAE),

[0058] 5-(4-nitrobenzoyl)aminosalicylic acid(NBAA),

[0059] 5-(4-nitrobenzenesulfonyl)aminosalicylic acid(NBSAA),

[0060] 5-[2-(4-nitrophenyl)-ethyl]aminosalicylic acid(NPAA), and

[0061] 5-[3-(4-nitrophenyl)-n-propyl]aminosalicylic acid(NPPAA), or apharmaceutically-acceptable salt thereof.

[0062] The term “pharmaceutically-acceptable salts” embraces saltscommonly used to form alkali metal salts and to form addition salts offree acids or free bases. The nature of the salt is not critical,provided that it is pharmaceutically acceptable, and acids or baseswhich may be employed to form such salts are, of course, well known tothose skilled in the art. Examples of acids which may be employed toform pharmaceutically acceptable acid addition salts include suchinorganic acids as hydrochloric acid, sulfuric acid and phosphoric acidand such organic acids as maleic acid, succinic acid and citric acid.Other salts include salts with alkali metals or alkaline earth metals,such as sodium, potassium, calcium and magnesium, or with organic bases,such as dicyclohexylamine. All of these salts may be prepared byconventional means from the corresponding compound of Formula (I) byreacting, for example, the appropriate acid or base with the compound ofFormula (I).

[0063] The Synthesis Examples show the exemplary method for thepreparation of the representative compounds (I).

SYNTHESIS EXAMPLE 1 Preparation of 5-benzylaminosalicylic acid (BAS)

[0064] To a solution of 5-aminosalicylic acid (2.0 g, 13 mmole,purchased from Aldrich Chemical Company, USA) and triethylamine in driedDMF (25 ml) was added benzyl bromide (2.68 g, 1.90 ml, 15.6 mmole) atroom temperature under a nitrogen atmosphere. The reaction mixture wasstirred for 4 hr at room temperature. Ice chips were added to thereaction mixture and then solvent was removed in vacuo. The reactionmixture was diluted with water and then extracted with ethyl acetate.The organic layer was washed with water and brine, and then dried overanhydrous MgSO₄. After evaporation of the solvent, the residue waspurified by column chromatography and recrystallized from methanol/ethylacetate/hexane (1:3:1) to give 3.6 g (73% yield) of 5-benzylaminosalylicacid as a white solid. : mp 173.5-174.5° C. (decompose).

[0065] Elemental analysis for C₁₄H₁₃NO₃. % C % H % N Calculated 69.125.39 5.76 Found 69.30 5.18 5.63

SYNTHESIS EXAMPLE 2 Preparation of 5-(4-nitrobenzyl)aminosalicylic acid(NBAS)

[0066] By following the similar procedure in Synthesis Example 1 byusing 5-aminosalicylic acid (2.00 g, 13.0 mmole) and 4-nitrobenzylbromide (3.38 g, 15.6 mmole), 2.90 g (79% yield) of5-(4-nitrobenzyl)aminosalicylic acid was obtained as a pale yellowsolid.: mp 211-212° C.

[0067] Elemental analysis for C₁₄H₁₃N₂O₅. % C % H % N Calculated 58.334.20 9.72 Found 58.38 4.21 9.71

SYNTHESIS EXAMPLE 3 Preparation of 5-(4-chlorobenzyl)aminosalicylic acid(CBAS)

[0068] By following the similar procedure in Synthesis Example 1 byusing 5-aminosalicylic acid (500 mg, 3.26 mmole) and 4-chlorobenzylchloride (630 mg, 3.91 mmole), 480 mg (53% yield) of5-(4-chlorobenzyl)aminosalicylic acid was obtained as a white solid.: mp227-228° C.

[0069] Elemental analysis for C₁₄H₁₂ClNO₃. % C % H % N Calculated 60.554.36 5.04 Found 60.43 4.21 5.02

SYNTHESIS EXAMPLE 4 Preparation of5-(4-trifluoromethylbenzyl)aminosalicylic acid (TBAS)

[0070] By following the similar procedure in Synthesis Example 1 byusing 5-aminosalicylic acid (500 mg, 3.26 mmole) and4-(trifluoromethyl)benzyl chloride (760 mg, 3.92 mmole), 510 mg (50%yield) of 5-(4-(trifluoromethyl)benzyl)aminosalicylic acid was obtainedas a white solid.: mp>188° C. (decompose).

[0071] Elemental analysis for C₁₄H₁₂F₃NO₃. % C % H % N Calculated 57.883.89 4.50 Found 57.61 3.98 4.44

SYNTHESIS EXAMPLE 5 Preparation of 5-(4-fluorobenzyl)aminosalicylic acid(FBAS)

[0072] By following the similar procedure in Synthesis Example 1 byusing 5-aminosalicylic acid (500 mg, 3.26 mmole) and 4-fluorobenzylbromide (740 mg, 3.92 mmole), 480 mg (44% yield) of5-(4-fluorobenzyl)aminosalicylic acid was obtained as a white solid.:mp>210° C. (decompose).

[0073] Elemental analysis for C₁₄H₁₂FNO₃. % C % H % N Calculated 64.364.63 5.36 Found 64.10 4.42 5.07

SYNTHESIS EXAMPLE 6 Preparation of 5-(4-methoxybenzyl)aminosalicylicacid (MBAS)

[0074] By following the similar procedure in Synthesis Example 1 byusing 5-aminosalicylic acid (1.00 g, 6.53 mmole) and 4-methoxybenzylchloride (1.23 g, 7.84 mmole), 890 mg (50% yield) of5-(4-methoxybenzyl)aminosalicylic acid was obtained as a white solid.:mp 205-206° C.

[0075] Elemental analysis for C₁₅H₁₅NO₄. % C % H % N Calculated 65.925.53 5.13 Found 65.83 5.45 5.07

SYNTHESIS EXAMPLE 7 Preparation of 5-(pentafluorobenzyl)aminosalicylicacid

[0076] By following the similar procedure in Synthesis Example 1 byusing 5-aminosalicylic acid (500 mg, 3.26 mmole) and pentafluorobenzylbromide (1.02 g, 3.92 mmole), 650 mg (60% yield) of5-(pentafluorobenzyl)aminosalicylic acid was obtained as a white solid.:mp>190° C. (decompose).

[0077] Elemental analysis for C₁₄H₈F₅NO₃. % C % H % N Calculated 50.462.42 4.20 Found 50.53 2.19 4.23

SYNTHESIS EXAMPLE 8 Preparation of 5-(4-nitrobenzyl)amino-2-hydroxyethylbenzoate

[0078] To a solution of 5-(4-nitrobenzyl)aminosalicylic acid (1.0 g, 3.4mmole) in ethanol (35 ml) was carefully added Conc.H₂SO₄ (3.5 ml) at 0°C. The reaction mixture was stirred for 6 hr at 80° C. and cooled toroom temperature. After the solvent was removed in vacuo, the reactionmixture was extracted with ethyl acetate. The organic layer was washedwith H₂O, 10% NaHCO₃ solution, 5% HCl solution and brine. After theorganic layer was dried over anhydrous MgSO₄, it was concentrated invacuo. The residue was purified by column chromatography andrecrystalized from ethyl acetate/hexane (1:2) to give 500 mg (46% yield)of 5-(4-nitrobenzyl)amino-2-hydroxy ethylbenzoate as a yellow solid.: mp106.5-107.5° C.

[0079] Elemental analysis for C₁₆H₁₆N₂O₅. % C % H % N Calculated 60.755.10 8.86 Found 60.68 5.24 9.04

SYNTHESIS EXAMPLE 9 Preparation of5-(4-nitrobenzyl)-N-acetylamino-2-hydroxy ethylbenzoate

[0080] To a solution of 5-(4-nitrobenzyl)amino-2-hydroxy ethylbenzoate(500 mg, 1.58 mmole) in dried methanol (50 ml) was carefully addedacetic anhydride (5 ml) at 0° C. under a nitrogen atmosphere. Thereaction mixture was stirred for 2 hr at 10° C. After ice chips wereslowly added to the reaction mixture, the solvent was removed in vacuo.The reaction mixture was extracted with ethyl acetate and H₂O, and theorganic layer was washed with H₂O, 10% NaHCO₃ (30 ml×3) solution, 5% HCl(30 ml×2) solution and brine. The organic solution was dried overanhydrous MgSO₄ and evaporated. The residue was purified by columnchromatography and recrystalized from ethyl acetate/hexane (1:3) to give400 mg (70% yield) of 5-(4-nitrobenzyl)-N-acetylamino-2-hydroxyethylbenzoate as a pale yellow solid. : mp 105.5-106.0° C.

[0081] Elemental analysis for C₁₈H₁₈N₂O₆. % C % H % N Calculated 60.335.06 7.82 Found 60.54 5.35 8.12

SYNTHESIS EXAMPLE 10 Preparation of5-(4-Nitrobenzyl)-N-acetylamino-2-acetoxy ethylbenzoate

[0082] To a solution of 5-(4-nitrobenzyl)amino-2-hydroxy ethylbenzoate(500 mg, 1.58 mmole) in acetic anhydride (10 ml) was carefully addedconc.H₂SO₄ (0.5 ml) at 0° C. under a nitrogen atmosphere. The reactionmixture was stirred for 30 min at 10° C. After ice chips were slowlyadded to the reaction mixture, the solvent was removed in vacuo. Thereaction mixture was diluted with water and extracted with ethylacetate. The organic layer was washed H₂O, 10% NaHCO₃ (30ml×3) solution,5% HCl (30 ml×2) solution and brine and then dried over anhydrous MgSO₄.After evaporation of the solvent, the residue was purified by columnchromatography to give 450 mg (71% yield) of5-(4-nitrobenzyl)-N-acetylamino-2-acetoxy ethylbenzoate as pale yellowoil.

[0083] Elemental analysis for C₂₀H₂₀N₂O₇. % C % H % N Calculated 60.005.03 7.00 Found 59.96 4.84 7.15

SYNTHESIS EXAMPLE 11 Preparation of 5-(4-nitrobenzoyl)aminosalicylicacid

[0084] By following the similar procedure in Synthesis Example I byusing 5-aminosalicylic acid (500 mg, 3.26 mmole) and 4-nitrobenzoylchloride (700 mg, 3.77 mmole), 550 mg (56% yield) of5-(4-nitrobenzoyl)aminosalicylic acid was obtained as a pale yellowsolid.: mp 270-271° C.

[0085] Elemental analysis for C₁₄H₁₀N₂O₆. % C % H % N Calculated 55.633.33 9.27 Found 55.82 3.43 9.08

SYNTHESIS EXAMPLE 12 Preparation of5-(4-nitrobenzenesulfonyl)aminosalicylic acid

[0086] By following the similar procedure in Synthesis Example 1 byusing 5-aminosalicylic acid (500 mg, 3.26 mmole) and4-nitrobenzenesulsonyl chloride (720 mg, 3.26 mmole), 390 mg (35% yield)of 5-(4-nitrobenzenesulfonyl)aminosalicylic acid was obtained as ayellow solid.: mp 239-240° C.

[0087] Elemental analysis for C₁₃H₁₀N₂O₇S. % C % H % N % S Calculated46.15 2.98 8.28 9.48 Found 46.27 2.92 8.34 9.51

SYNTHESIS EXAMPLE 13 Preparation of5-[2-(4-nitrophenyl)ethyl]aminosalicylic acid

[0088] By following the similar procedure in Synthesis Example 1 byusing 5-aminosalicylic acid (500 mg, 3.26 mmole) and 4-nitrophenethylbromide (900 mg, 3.92 mmole), 890 mg (50% yield) of5-(4-nitrophenethyl)aminosalicylic acid was obtained as a pale yellowsolid. : mp 234-236° C.

[0089] Elemental analysis for C₁₅H₁₄N₂O₅. % C % H % N Calculated 59.604.67 9.27 Found 59.77 4.79 9.24

SYNTHESIS EXAMPLE 14 Preparation of5-[3-(4-nitrophenyl)-n-propyl]aminosalicylic acid

[0090] By following the similar procedure in Synthesis Example 1 byusing 5-aminosalicylic acid (500 mg, 3.26 mmole) and3-(4-nitrophenyl)propyl bromide (950 mg, 3.92 mmole), 520 mg (50% yield)of 5-[3-(4-nitrophenyl)-n-propyl]aminosalicylic acid was obtained as apale yellow solid.: mp 229-231° C.

[0091] Elemental analysis for C₁₆H₁₆N₂O₅. % C % H % N Calculated 60.755.10 8.86 Found 60.77 5.07 8.89

EXPERIMENTAL EXAMPLE

[0092] Primary cortical cell cultures from embryonic mice were preparedand used to examine neuroprotective action of compounds. Mouse corticalcell culture system has been extensively used to study mechanisms andpharmacological intervention of neuronal death in neurological diseases.In brief, mouse cerebral cortices were removed from brains of the 15day-old-fetal mice, in accordance with a protocol approved by ourinstitutional animal care committee. The neocortices were gentlytriturated and plated on 24 well plates (5 hemispheres/plate) precoatedwith 100 μg/ml poly-D-lysine and 4 μg/ml laminine. Plating media consistof Eagles minimal essential media (MEM, Earles salts, suppliedglutamine-free) supplemented with 5% horse serum, 5% fetal bovine serum,2 mM glutamine, and 21 mM glucose. Cultures were maintained at 37° C. ina humidified 5% CO₂ atmosphere. After 7 days in vitro (DIV 7), cultureswere shifted into a growth medium identical to the plating medium butlacking fetal serum. At DIV 7-9, 10 mM cytosine arabinofuranoside wasincluded to halt overgrowth of glia. Mixed cultures of neurons and gliawere then fed twice a week.

[0093] To induce neuronal injury by NMDA or Zn²⁺, cortical cell cultureswere exposed to toxic doses of NMDA for 10 min or Zn²⁺ for 30 min in aHEPES-buffered control salt solution (HCSS): (in mM) 120 NaCl, 5KCl, 1.6MgCl₂, 2.3 CaCl₂, 15 glucose, 20 HEPES and 10 NaOH. After exposure,cultures were washed out 3 times and exchanged with MEM adjusted to 25mM glucose and 26.2 mM sodium bicarbonate, and placed in the CO₂incubator for the next 20-24 hr. To induce free radical neurotoxicity,cortical cell cultures were continuously exposed to Fe²⁺ or buthioninesulfoximine (BSO) for 20-24 hr, in MEM adjusted to 25 mM glucose and26.2 mM sodium bicarbonate.

[0094] Overall cell injury was assessed microscopically underphase-contrast optics or by measuring amount of lactate dehydrogenase(LDH) released into the bathing medium 24 hr after neurotoxic insults aspreviously described (Koh and Choi, J Neurosci Methods 20:83-90, 1987).The percent neuronal death was normalized to the mean LDH value released24 hr after continuous exposure 500 μM NMDA (=100) or a sham control(=0).

[0095] To examine anti-oxidant effect, DPPH(2,2-diphenyl-1-picryl-hydrazyl radical), a stable free radical, wasdissolved in ethanol to make a 100 μM solution. A compound was reactedwith DPPH or ethanol. After incubation for 30 min, relative decrease inDPPH absorption at 517 nm was measured by a spectrophotometer.

EXPERIMENTAL EXAMPLE 11 Anti-oxidant action of 5- amino salicylic acid

[0096] The neuroprotective action of 5-amino salicylic acid (AS) wasexamined in primary cortical cell cultures. Mouse cortical cell cultures(DIV 12-14) were exposed to 300 μM NMDA for 10 min (1 a), continuouslyto 50 μM Fe²⁺ (1 b), or 300 μM Zn²⁺ for 30 min (1 c), alone or withinclusion of 10-300 μM 5-amino salicylic acid (AS). Neuronal death wasanalyzed 24 hr later by measuring levels of LDH released into thebathing medium, mean ±SEM (n=9-12 (1 a), n=4-8 (1 b) or n=4 (1 c)culture wells per condition), scaled to mean LDH efflux value 24 hrafter sham wash (=0) and continuous exposure to 500 μM NMDA (=100). Theresult as shown in FIG. 1 indicates significant difference from relevantcontrol at p<0.05 using ANOVA and Student-Neuman-Keuls' test. Inclusionof 10-300 μM AS did not attenuate neuronal death evolving 24 hr after 10min-exposure to 300 μM NMDA (FIG. 1a). Interestingly, addition of 10-100μM AS dose-dependently prevented free radical neurotoxicity following 24hr-exposure to Fe²⁺ (FIG. 1b). Neuronal death 24 hr after 30min-exposure to 300 μM Zn²⁺ was not reduced by continuous inclusion ofAS during and post Zn²⁺ treatment (FIG. 1c). The neuroprotective actionof AS against free radical neurotoxicity was attributable to directanti-oxidant property of the compound as AS decreased levels of DPPH, astable free radical (Table 1). Compared to trolox, a membrane-permeableform of vitamin E, AS was a weak anti-oxidant. TABLE 1 Anti-oxidantproperty of AS Concentrations of Trolox or AS (μM) Reactants 0 3 10 30100 300 A_(571 nm) DPPH alone 1.2 ± 0.05 DPPH + Trolox 1.08 ± 0.1 0.89 ±0.08* 0.39 ± 0.06* 0.05 ± 0.01* 0.03 ± 0.01* DPPH + AS — 1.03 ± 0.05  0.9 ± 0.06* 0.45 ± 0.07* 0.16 ± 0.00*

[0097] AS or trolox was reacted with 100 uM DPPH dissolved in ethanolfor 30 min. Anti-oxidant property was analyzed by measuring changes inthe level of DPPH at 517 nm, mean ±SEM (n=3 test tubes per condition),after subtracting background value resulting from ethanol alone. FIG. 1indicates significant difference from DPPH alone at P<0.05, using ANOVAand Student-Neuman-Keuls test.

[0098] AS can protect neurons from free radical injuries withoutbeneficial effects against NMDA or Zn²⁺ neurotoxicity. However, AS isweaker than trolox in scavenging free radicals.

EXPERIMENTAL EXAMPLE 2 Neuroprotective Effects of 5-benzylaminosalicylicacid and its Derivatives 1. Neuroprotective Effects of5-benzylaminosalicylic acid

[0099] BAS was synthesized and examined against neuronal injuriesinduced in cortical cell cultures. Mouse cortical cell cultures (DIV12-14) were exposed to 300 μM NMDA for 10 min (2 a), continuously to 50μM Fe²⁺ (2 b) or 10 mM BSO (2 c), or 300 μM Zn²⁺ for 30 min (2 d), aloneor with inclusion of indicated doses of 5-benzylamino salicylate (BAS).Neuronal death was analyzed 24 hr later by measuring levels of LDHreleased into the medium, mean ±SEM (n=7-8 (2 a), n=3-6 (2 b), n=4 (2c), or n=4 (2 d) culture wells per condition). FIG. 2. indicatessignificant difference from relevant control, at p<0.05 using ANOVA andStudent-Neuman-Keuls test. Concurrent addition of 300 μM5-benzylaminosalicylic acid (BAS) reduced NMDA-induced neuronal deathapproximately by 50% (FIG. 2a). Neuronal death following exposure to 50μM Fe²⁺ (FIG. 2b) or 10 mM BSO (FIG. 2c) was substantially reduced inthe presence of 1 μM BAS and near completely blocked by addition of 3 μMBAS. Administration of 30-300 μM BAS dose-dependently reduced neuronaldeath 24 hr following exposure to 300 μM Zn²⁺ for 30 min (FIG. 2d). LikeAS or trolox, BAS acted as a direct anti-oxidant (Table 2). Theanti-oxidant property of BAS was observed at a dose as low as 1 μM.TABLE 2 Anti-oxidant property of BAS [BAS], μM 0 1 3 10 30 100 300A_(571 nm) 1.2 ± 0.05 1.07 ± 0.07 1.01 ± 0.07 0.71 ± 0.09* 0.21 ± 0.04*0.15 ± 0.02* 0.16 ± 0.01*

[0100] BAS was reacted with 100 uM DPPH dissolved in ethanol for 30 min.Anti-oxidant property was analyzed by measuring changes in DPPH at 517nm, mean ±SEM (n=3 test tubes per condition), after subtractingbackground value resulting from ethanol alone. FIG. 1 indicatessignificant difference from DPPH alone ([BAS]=O) at P<0.05, using ANOVAand Student-Neuman-Keuls test.

2. BAS Derivatives

[0101] BAS derivatives were synthesized by substituting —H at paraposition of benzylamino group with —NO₂[5-(4-nitrobenzylamino)salicylicacid, NBAS], —Cl[5-(4-chlorobenzylamino)salicylic acid, CBAS],—CF₃[5-(4-trifluoromethylbenzylamino)salicylic acid, TBAS]. Mousecortical cell cultures (DIV 12-14) were exposed to 300 μM NMDA for 10min (3 a), continuously to 50 μM Fe²⁺ (3 b), or 300 μM Zn²⁺ for 30 min(3 c), alone or with inclusion of indicated doses of5-(4-nitrobenzyl)aminosalicylic acid (NBAS). Neuronal death was analyzed24 hr later by measuring levels of LDH released into the medium, mean±SEM (n=7-8 (3 a), n=3-6 (3 b), or n=4 (3 c) culture wells percondition). The result as shown in FIG. 3 indicates significantdifference from relevant control, at p<0.05 using ANOVA andStudent-Neuman-Keuls test.

[0102] Mouse cortical cell cultures (DIV 12-14) were exposed to 300 μMNMDA for 10 min (4 a), continuously to 50 μM Fe²⁺ (4 b), or 300 μM Zn²⁺for 30 min (4 c), alone or with inclusion of indicated doses of5-(4-chlorobenzyl)aminosalicylic acid (CBAS). Neuronal death wasanalyzed 24 hr later by measuring levels of LDH released into themedium, mean ±SEM (n=3-4 (4 a), n=3-12 (4 b), or n=4 (4 c) culture wellsper condition). The result as shown in FIG. 4 indicates significantdifference from relevant control, at p<0.05 using ANOVA andStudent-Neuman-Keuls test.

[0103] Mouse cortical cell cultures (DIV 12-14) were exposed to 300 μMNMDA for 10 min (5 a), continuously to 50 μM Fe²⁺ (5 b), or 300 μM Zn²⁺for 30 min (5 c), alone or with inclusion of indicated doses of5-(4-Trifluoromethylbenzyl)aminosalicylic acid (TBAS). Neuronal deathwas analyzed 24 hr later by measuring levels of LDH released into themedium, mean ±SEM (n=3-4 (5 a), n=3-11 (5 b), or n=4 (5 c) culture wellsper condition). The result as shown in FIG. 5 indicates significantdifference from relevant control, at p<0.05 using ANOVA andStudent-Neuman-Keuls test.

[0104] Mouse cortical cell cultures (DIV 12-14) were exposed to 300 μMNMDA for 10 min (6 a) or continuously to 50 μM Fe²⁺ (6 b) alone or withinclusion of indicated doses of 5-(4-Fluorobenzyl)aminosalicylicacid(FBAS). Neuronal death was analyzed 24 hr later by measuring levelsof LDH released into the medium, mean ±SEM (n=7-8 (6 a) or n=4-8 (6 b)culture wells per condition). The result as shown in FIG. 6 indicatessignificant difference from relevant control, at p<0.05 using ANOVA andStudent-Neuman-Keuls test.

[0105] Mouse cortical cell cultures (DIV 12-14) were exposed to 300 μMNMDA for 10 min (7 a) or continuously to 50 μM Fe²⁺ (7 b) alone or withinclusion of indicated doses of 5-(4-methoxybenzyl)aminosalicylic acid(MBAS). Neuronal death was analyzed 24 hr later by measuring levels ofLDH released into the medium, mean ±SEM (n=7-8 (7 a) or n=4-8 (7 b)culture wells per condition). The result as shown in FIG. 7 indicatessignificant difference from relevant control, at p<0.05 using ANOVA andStudent-Neuman-Keuls test.

[0106] This substitution with electron-withdrawing group did not reduceneuroprotective effects of BAS against NMDA, Zn²⁺, or free radicalneurotoxicity (FIGS. 3-5). These BAS derivatives were more potent thanBAS in preventing free radical neurotoxicity. TABLE 3 Anti-oxidantproperty of NBAS [NBAS],μM 0 10 30 100 300 A_(571 nm) 1.2 ± 0.01 0.37 ±0.1* 0.04 ± 0.01* 0.04 ± 0.00* 0.04 ± 0.00*

[0107] NBAS was reacted with 100 uM DPPH dissolved in ethanol for 30min. Anti-oxidant property was analyzed by measuring changes in DPPH at517 nm, mean ±SEM (n=3 test tubes per condition), after subtractingbackground value resulting from ethanol alone. Table 3 indicatessignificant difference from DPPH alone ([reactant]=O) at P<0.05, usingANOVA and Student-Neuman-Keuls test.

[0108] Substituting —NO₂ with —F or —OCH₃ resulted in decreasedneuroprotection against NMDA toxicity but appeared to increaseneuroprotective potential against free radical injury (FIGS. 6 and 7).

[0109] EXPERIMENTAL EXAMPLE 3

Neuroprotective effects of 5-(Pentafluorobenzyl)amino salicylic acid

[0110] 5-(pentafluorobenzyl)amino salicylic acid (PBAS) was synthesizedand tested against neuronal injuries. Mouse cortical cell cultures (DIV12-14) were exposed to 300 μM NMDA for 10 min (8 a), continuously to 50μM Fe²⁺ (8 b) or 10 mM BSO (8 c), or 300 μM Zn²⁺ for 30 min (8 d), aloneor with inclusion of indicated doses of 5-(pentafluorobenzyl)aminosalicylic acid (PBAS). Neuronal death was analyzed 24 hr later bymeasuring levels of LDH released into the medium, mean ±SEM (n=11-16 (8a), n=3-6 (8 b), n=4-11 (8 c), or n=12 (8 d) culture wells percondition). The result as shown in FIG. 8 indicates significantdifference from relevant control, at p<0.05 using ANOVA andStudent-Neuman-Keuls test. Concurrent addition of 100-1000 μM PBASreduced NMDA-induced neuronal death in a dose-dependent manner.Treatment with 300 μM PBAS reduced NMDA neurotoxicity approximately by65% (FIG. 8a). Increasing doses of PBAS up to 1 mM completely blockedneuronal death following exposure to 300 μM NMDA. Inclusion of 1 μM PBASsignificantly reduced neuronal death following continuous exposure to 50μM Fe²⁺. Fe²⁺-induced neuronal death was near completely blocked in thepresence of 3 μM PBAS. Free radical neurotoxicity resulting fromexposure to 10 mM BSO was blocked by addition of 1 μM PBAS. PBAS reducedlevels of DPPH (table 4), suggesting that PBAS blocked free radicalneurotoxicity as a direct antioxidant. Concurrent addition of 100-300 μMPBAS attenuated Zn²⁺ neurotoxicity (FIG. 8d). TABLE 4 Anti-oxidantproperty of PBAS [PBAS],μM 0 1 3 10 30 100 300 A_(571 nm) 1.2 ± 0.011.07 ± 0.07 1.01 ± 0.07 0.71 ± 0.09* 0.21 ± 0.04* 0.15 ± 0.02* 0.16 ±0.01*

[0111] PBAS was reacted with 100 uM DPPH dissolved in ethanol for 30min. Anti-oxidant property was analyzed by measuring changes in DPPH at517 nm, mean ±SEM (n=3 test tubes per condition), after subtractingbackground value resulting from ethanol alone. Table 4indicatessignificant difference from DPPH alone ([PBAS]=0) at P<0.05, using ANOVAand Student-Neuman-Keuls test.

EXPERIMENTAL EXAMPLE 4 Neuroprotective Effects of NBAS Derivatives(X═CH₂)

[0112] Several derivatives of PBAS such as5-(4-nitrobenzyl)amino-2-hydroxy ethylbenzoate (NAHE; R₁═H, R₂═CH₂CH₃,R₃═H), 5-(4-nitrobenzyl)-N-acetylamino-2-hydroxy ethylbenzoate (NNAHE;R₁═COCH₃, R₂═CH₂CH₃, R₃═H), and5-(4-nitrobenzyl)-N-acetylamino-2-acetoxy ethylbenzoate (NNAAE;R₁═COCH₃, R₂═CH₂CH₃, R₃═COCH₃) were synthesized and theirneuroprotection action was examined in cortical cell cultures. Mousecortical cell cultures (DIV 12-14) were exposed to 300 μM NMDA for 10min (9 a) or continuously to 50 μM Fe²⁺ (9 b), alone or with inclusionof indicated doses of ethyl-5-(4-nitrobenzyl)amino-2-hydroxyethylbenzoate (NAHE). Neuronal death was analyzed 24 hr later bymeasuring levels of LDH released into the medium, mean ±SEM (n=10-12 (9a) or n=3-4 (9 b) culture wells per condition). The result as shown inFIG. 9 indicates significant difference from relevant control, at p<0.05using ANOVA and Student-Neuman-Keuls test.

[0113] Mouse cortical cell cultures (DIV 12-14) were exposed to 300 μMNMDA for 10 min (10 a) or continuously to 50 μM Fe²⁺ (10 b) alone orwith inclusion of indicated doses of5-(4-nitrobenzyl)-N-acetylamino-2-hydroxy ethylbenzoate (NNAHE).Neuronal death was analyzed 24 hr later by measuring levels of LDHreleased into the medium, mean ±SEM (n=3-4 (10 a) or n=3-4 (10 b)culture wells per condition). The result as shown in FIG. 10 indicatessignificant difference from relevant control, at p<0.05 using ANOVA andStudent-Neuman-Keuls test.

[0114] Mouse cortical cell cultures (DIV 12-14) were exposed to 300 μMNMDA for 10 min (11 a) or continuously to 50 μM Fe²⁺ (11 b), alone orwith inclusion of indicated doses of5-(4-nitrobenzyl)-N-acetylamino-2-acetoxy ethylbenzoate (NNAAE).Neuronal death was analyzed 24 hr later by measuring levels of LDHreleased into the medium, mean ±SEM (n=10-12 (11 a), n=7-8 (11 b), orn=4 (11 c) culture wells per condition). The result as shown in FIG. 11indicates significant difference from relevant control, at p<0.05 usingANOVA and Student-Neuman-Keuls test. These derivatives attenuated NMDAneurotoxicity at 300 μM (FIGS. 9-11). Inclusion of 10 μM NAHE, NNAHE, orNNAAE blocked Fe²⁺-induced free radical neurotoxicity.

EXPERIMENTAL EXAMPLE 5 Neuroprotective Effects of NBAS Derivatives(X═CO, SO₂, CH₂CH₂ or CH₂CH₂CH₂)

[0115] The group of X (CH₂) of NBAS was substituted with CO[5-(4-nitrobenzoyl)aminosalicylic acid; NBAA],SO₂[5-(4-nitrobenzenesulfonyl)aminosalicylic acid; NBSAA], CH₂CH₂[5-(4-nitrophenethyl)aminosalicylic acid; NPAA], orCH₂CH₂CH₂[5-[3-(4-nitrophenyl)-n-propyl]aminosalicylic acid; NPPAA].Mouse cortical cell cultures (DIV 12-14) were exposed to 300 μM NMDA for10 min (12 a) or continuously to 50 μM Fe²⁺ (12 b), alone or withinclusion of indicated doses of 5-(4-nitrobenzonyl)aminosalicylicacid(NBAA). Neuronal death was analyzed 24 hr later by measuring levelsof LDH released into the medium, mean ±SEM (n=7-8 (12 a) or n=3-4 (12 b)culture wells per condition). The result as shown in FIG. 12 indicatessignificant difference from relevant control, at p<0.05 using ANOVA andStudent-Neuman-Keuls test.

[0116] Mouse cortical cell cultures (DIV 12-14) were exposed to 300 μMNMDA for 10 min (13 a) or continuously to 50 μM Fe²⁺ (13 b) alone orwith inclusion of indicated doses of5-(4-nitrobenzenesulfonyl)aminosalicylic acid(NBSAA). Neuronal death wasanalyzed 24 hr later by measuring levels of LDH released into themedium, mean ±SEM (n=3-4 (13 a) or n=2-8 (13 b) culture wells percondition). The result as shown in FIG. 13 indicates significantdifference from relevant control, at p<0.05 using ANOVA andStudent-Neuman-Keuls test.

[0117] Mouse cortical cell cultures (DIV 12-14) were exposed to 300 μMNMDA for 10 min (14 a) or continuously to 50 μM Fe²⁺ (14 b), alone orwith inclusion of indicated doses of5-[2-(4-nitrophenyl)-ethyl]aminosalicylic acid(NPAA). Neuronal death wasanalyzed 24 hr later by measuring levels of LDH released into themedium, mean ±SEM (n=4 (14 a) or n=4-8 (14 b) culture wells percondition). The result as shown in FIG. 14 indicates significantdifference from relevant control, at p<0.05 using ANOVA andStudent-Neuman-Keuls test.

[0118] Mouse cortical cell cultures (DIV 12-14) were exposed to 300 μMNMDA for 10 min (15 a) or continuously to 50 μM Fe²⁺ (15 b), alone orwith inclusion of indicated doses of5-[3-(4-nitrophenyl)-n-propyl]aminosalicylic acid (NPPAA). Neuronaldeath was analyzed 24 hr later by measuring levels of LDH released intothe medium, mean ±SEM (n=4 (15 a) or n=3-8 (15 b) culture wells percondition). The result as shown in FIG. 15 indicates significantdifference from relevant control, at p<0.05 using ANOVA andStudent-Neuman-Keuls test. NBAA at a dose of 30 μM significantlyattenuated NMDA neurotoxicity. However, its protective effect againstNMDA was not further increased up to doses of 300 μM (FIG. 12a).Interestingly, inclusion of 30-300 μM NBSAA attenuated NMDAneurotoxicity in a dose-dependent manner (FIG. 13a). With inclusion of300 μM NBSAA, 90-100% neuronal death following exposure to 300 μM NMDAwas markedly reduced. NBAA and NBSAA were still neuroprotective againstFe²⁺ injury but weaker than NBAS in reducing free radical neurotoxicity(FIGS. 12 and 13; Table 5).

[0119] Substitution of CH₂ for X with CH₂CH₂ or CH₂CH₂CH₂ reducedprotective potency of NBAS against NMDA neurotoxicity. Administration of300 μM NPAA or NPPAA slightly reduced NMDA-induced neuronal death incortical neurons (FIGS. 14 and 15). In contrast, NPAA or NPPAA turnedout to be more effective than NBAS in blocking free radicalneurotoxicity as shown by complete blockade of Fe²⁺-induced neuronaldeath in the presence of 1 μM NPAA or NPPAA. TABLE 5 Anti-oxidantproperty of NBAS derivatives Reactants DPPH alone DPPH + NBAA DPPH +NBSAA DPPH + NPAA DPPH + NPPAA A_(571 nm) 1.2 ± 0.01 0.42 ± 0.22* 0.15 ±0.05* 0.09 ± 0.01* 0.09 ± 0.02*

[0120] NBAS derivatives (100 μM for each) were reacted with 100 μM DPPHdissolved in ethanol for 30 min. Anti-oxidant property was analyzed bymeasuring changes in DPPH at 517 nm, mean ±SEM (n=3 test tubes percondition), after subtracting background value resulting from ethanolalone. Table 5 indicates significant difference from DPPH alone atP<0.05, using ANOVA and Student-Neuman-Keuls test.

[0121] The above results show that the compounds of Formula (I) may beemployed efficiently to prevent neurodegenerative diseases inassociation with excitotoxicity, Zn²⁺ neurotoxicity and free radicalneurotoxicity.

[0122] Administration of compounds within Formula (I) to humans can beby any technique capable of introducing the compounds into thebloodstream of a human patient or mammals, including oraladministration, and by intravenous, intramuscular and subcutaneousinjections The mammals include, but not limited to, cats, dogs, poultry,cattle and the like.

[0123] Compounds indicated for prophylactic therapy will preferably beadministered in a daily dose generally in a range from about 0.1 mg toabout 20 mg per kilogram of body weight per day. A more preferred dosagewill be a range from about 0.2 mg to about 10 mg per kilogram of bodyweight. Most preferred is a dosage in a range from about 0.5 to about 5mg per kilogram of body weight per day. A suitable dose can beadministered, in multiple sub-doses per day. These sub-doses may beadministered in unit dosage forms. Typically, a dose or sub-dose maycontain from about 1 mg to about 100 mg of active compound per unitdosage form. A more preferred dosage will contain from about 2 mg toabout 50 mg of active compound per unit dosage form. Most preferred is adosage form containing from about 3 mg to about 25 mg of active compoundper unit dose.

[0124] The active compound is usually administered in apharmaceutically-acceptable formulation. Such formulations may comprisethe active compound together with one or morepharmaceutically-acceptable carriers or diluents. Other therapeuticagents may also be present in the formulation. Apharmaceutically-acceptable carrier or diluent provides an appropriatevehicle for delivery of the active compound without introducingundesirable side effects. Delivery of the active compound in suchformulations may be performed by various routes including oral, nasal,topical, buccal and sublingual, or by parenteral administration such assubcutaneous, intramuscular, intravenous and intradermal routes.

[0125] Formulations for oral administration may be in the form ofcapsules containing the active compound dispersed in a binder such asgelatin or hydroxypropylmethyl cellulose, together with one or more of alubricant, preservative, surface-active or dispersing agent. Suchcapsules or tablets may contain controlled-release formulation as may beprovided in a disposition of active compound in hydroxypropylmethylcellulose.

[0126] Formulations for parenteral administration may be in the form ofaqueous or non-aqueous isotonic sterile injection solutions orsuspensions. These solutions and suspensions may be prepared fromsterile powders or granules having one or more of the carriers ordiluents mentioned for use in the formulations for oral administration.

[0127] Although this invention has been described with respect tospecific embodiments, the details of these embodiments are not to beconstrued as limitations. Various equivalents, changes and modificationsmay be made without departing from the spirit and scope of thisinvention, and it is understood that such equivalent embodiments arepart of this invention.

What is claimed is:
 1. A method for protecting central neurons fromacute or chronic injuries to the central nervous system (CNS) comprisingadministering to a patient or a mammal a therapeutically appropriateamount of one or more compounds represented by the following formula (I)or pharmaceutically acceptable salts thereof:

wherein, X is CO, SO₂ or (CH₂)n, wherein n is an integer of 1 to 5; R₁is hydrogen, alkyl or alkanoyl; R₂ is hydrogen or alkyl; R₃ is hydrogenor an acetoxy group; and R₄ is phenyl group which is unsubstituted orsubstituted with one or more of the group consisting of nitro, halogen,haloalkyl, and C₁-C₅ alkoxy.
 2. The method according to claim 1, whereinsaid CNS injuries result from ischemia, hypoxia, hypoglycemia, traumaticbrain injury, traumatic spinal cord injury, epilepsy, Huntingtonsdisease, Parkinsons disease, Alzheimer's disease, or Amytrophic lateralsclerosis.
 3. The method according to claim 1, wherein said CNS injuriesare caused by activation of N-methyl-D-aspartate (NMDA) glutamatereceptors, entry and accumulation of Zn²⁺, or free radicals.
 4. Themethod according to claim 1, wherein the compounds are selected from thegroup consisting of; 5-benzylaminosalicylic acid (BAS),5-(4-nitrobenzyl)aminosalicylic acid (NBAS),5-(4-chlorobenzyl)aminosalicylic acid (CBAS),5-(4-trifluoromethylbenzyl)aminosalicylic acid (TBAS),5-(4-fluorobenzyl)aminosalicylic acid (FBAS),5-(4-methoxybenzyl)aminosalicylic acid (MBAS)5-(4-pentafluorobenzyl)aminosalicylic acid (PBAS),5-(4-nitrobenzyl)amino-2-hydroxy ethylbenzoate,5-(4-nitrobenzyl)-N-acetylamino-2-hydroxy ethylbenzoate,5-(4-nitrobenzyl)-N-acetylamino-2-acetoxy ethylbenzoate,5-(4-nitrobenzoyl)aminosalicylic acid,5-(4-nitrobenzenesulfonyl)aminosalicylic acid,5-[2-(4-nitrophenyl)ethyl]aminosalicylic acid, and5-[3-(4-nitrophenyl)-n-propyl]aminosalicylic acid, or apharmaceutically-acceptable salt thereof.